Excited State Pathways Leading to Formation of
Adenine Dimers
Akos Banyasz, Lara Martinez-Fernandez, Tiia-Maaria Ketola, A. Munoz-Losa,
Luciana Esposito, Dimitra Markovitsi, Roberto Improta
To cite this version:
Akos Banyasz, Lara Martinez-Fernandez, Tiia-Maaria Ketola, A. Munoz-Losa, Luciana Esposito, et al.. Excited State Pathways Leading to Formation of Adenine Dimers. Journal of Physical Chemistry Letters, American Chemical Society, 2016, 7, pp.2020–2023.
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Excited State Pathways Leading to Formation of
Adenine Dimers
Akos Banyasz,a Lara Martinez-Fernandez,b Tiia-Maaria Ketola,a¥ Aurora Muñoz-Losa,a†
Luciana Esposito,b Dimitra Markovitsi*a and Roberto Improta*b
a
LIDYL, CEA, CNRS, Université Paris-Saclay, F-91191 Gif-sur-Yvette, France
b
Istituto Biostrutture e Bioimmagini- Consiglio Nazionale delle Ricerche, Via Mezzocannone
16, I-80134 Napoli, Italy
AUTHOR INFORMATION
Corresponding Authors
[email protected]
[email protected]
¥
present address: CP-Kelco Oy, Kuhnamontie 2, PL 500, FI-44101 Äänekoski, Finland.
present address: Institute of Theoretical Chemistry, University of Vienna, Währinger Str. 17,
1090 Vienna, Austria.
†
1
ABSTRACT
The reaction intermediate in the path leading to UV-induced formation of adenine dimers A=A
and AA* is identified for the first time quantum mechanically, using PCM/TD-DFT calculations
on (dA)2. In parallel, its fingerprint is detected in the absorption spectra recorded on the
millisecond time-scale for the single strand (dA)20.
2
The characterization of photochemical reactions of nucleic acids is important in
connection with the UV-induced damage to the genetic code. Although a large number of
publications deal with pyrimidine dimerization, very few focus on adenine dimer
formation.1-8 Yet, the photodimerization quantum yield of adenine single strands is the
order of 10-3,6 comparable to that of (6-4)pyrimidine-pyrimidone adducts (64PPs) in
thymine single strands,9 which have received considerable attention. 10Intriguingly, the
photodimerization yields dramatically drop in DNA duplexes and in RNA strands. 3,
11
6,
Experimental studies demonstrated that irradiation around 260 nm leads to the
formation of two different types of adenine dimers AA* and A=A (Fig. 1) and suggested
that they have a common azetidine intermediate. 5 Here, we report the first theoretical
study exploring the excited state pathways that lead to the azetidine precursor. The
fingerprint of the reaction intermediate, which, according to our calculations, presents
weak electronic transitions at longer wavelengths compared to AA* and A=A, is detected
in the absorption spectra recorded at 20 ms following 266 nm excitation by 5 ns laser
pulses of the single strand (dA)20.
Using extensive molecular dynamics (MD) analysis of (dA)5 (see SI, Fig. SI-1), we found
that a significant percentage of adenines undergoes syn/anti conformational equilibrium,
syn glycosidic conformers being particularly stable for the 5’-terminal base. Then, the
four (AA) dinucleotides corresponding to the different combinations of conformers,
namely (AA)anti-anti, (AA)syn-anti, (AA)anti-syn, and (AA)syn-syn, have been optimized at the
M052X/6-31G(d) level,12 including bulk solvent effects by means of the Polarizable
Continuum Model (PCM).13We have focused on C2’-endo/C2’-endo conformers, since
MD simulations predict that most of the ‘inner bases’ (those more representative of the
3
behavior in long strands) adopts a C2’-endo puckering. At the PCM/M052X level of
theory, the most stable conformer is (AA)syn-anti (Fig. SI-3 and Table SI-1), which is
stabilized by a strong hydrogen bond between the terminal CH2OH group and N3 atom of
the 5’-terminal adenine (5N3) (Fig. 1). When the molecules of the first solvation shell (see
SI, Fig. SI-2) are included, (AA)anti-antiis found to be the most stable conformer, but the
difference in energy with respect to (AA) syn-anti is only 4.0 kcal/mol, suggesting that a
very small, but non-zero, percentage of the AA steps adopts this conformation.
f-CI* (a) 5N3 5N9 fl (b) 5C2 5N1 5C4 5 C5 5C8 5C6 5N7 1.657 3
C5 5C8 5N7 3C5 3C6 1.581 2.498 1.528 M
M
(c) AA* (d) A=A d
p
Figure 1. Schematic drawing, atom labeling and selected bond distances (in Å) of relevant
structures in the photodimerization path of (AA)syn-anti conformers of (dA)n oligonucleotides: (a)
-CI*, corresponding to the conical intersection, (b) reaction intermediate I, (c) final photoproduct AA* and (d) final photo-product A=A.
Excited state PCM/TD-M052X calculations, which have been successfully applied to the
study of DNA photochemistry,9,
energy
excited
states
in
14-15
indicate that, for all conformers, the two lowest
the
Franck-Condon
region
correspond
to
symmetric/antisymmetric combinations (Frenkel excitons) of the lowest energy *
4
excited state of adenine (Table SI-2), denoted as La. Geometry optimization of the S1
excited state leads to minima (Fig. 2 and Fig. SI-3) characterized by a close approach of
the two bases. The S1 minimum found for (AA)syn-antiexhibits very short 5C8-3C5 and 5N73
C6 distances (the superscripts 5 and 3 correspond to the bases at the 5’ and 3’-end,
respectively). The HOMOLUMO electronic transition involves transfer of an electron
to an orbital with bonding character in respect to these atom pairs (Fig. 2). An extensive
analysis of the potential energy surfaces along the 5C8-3C5 coordinate shows that an
extremely low energy barrier (<0.08 eV) separates the S 1 minimum from a crossing region
with S0 in the path leading to photodimerization.
S1 5.36 4.92 4.84 f-CI* S1 Min ~4.2 FC LUMO 2.87 fl Figure 2. Schematic description of the potential energy surface in the path leading from the
Franck-Condon state to the minimum of the S 1 excited state and, then, to the formation of the
azetidine precursor I. PCM/TD-M052X/6-31G(d) calculations for the in (AA)syn-anti conformer.
The energy is given in eV.
In general, TD-DFT cannot provide an accurate description of conical intersections
However, in the case of several photodimerization processes in DNA, the description
obtained for the S1/S0 crossing region by TD-M052X is very similar to that obtained by
wavefunction based methods.9,
14-15
A representative structure corresponding to this
5
region (CI*), is shown in Fig. 1a. Subsequent S1 geometry optimization leads to the
photo-adduct I presented in Fig. 1b.
According to our calculations, the three other conformers do not exhibit any significant
tendency to photodimerization (see SI for details, Figs. SI-4, 5 and 6). The general picture
obtained for (AA)anti-synis qualitatively similar to that shown in Fig. 2 but the energy
barrier separating the S1 minimum from the crossing region is much higher (≥0.33 eV) . In
the S1 minimum of (AA)syn-syn the 5C8 and 3C8 atoms of adenines are very close (1.6 Å);
further approach leads to a crossing region with S0 and decay back to the ground state of
(AA)syn-syn. For (AA)anti-anti, the calculation along the 5C8-3C5 coordinate involves a large
increase in the energy (>0.5eV), making the access to I impossible. Finally, in the case
of (AA)anti-anti, we located another possible photo-adduct (see SI) where 5C5-3C5 and 5C63
C6 are bonded. However, the latter photo-adduct is less stable than I by 1.2 eV.
Therefore, I appears the only relevant intermediate in the path to A=A and AA* and
corresponds to the species postulated 25 years ago by Sharma and Davies.5
6
Figure 3. (A) Low energy electronic transitions of A=A and AA* dimers (black) and the
precursor I (red); PCM/TD-CAM-B3LYP/6-31+G(d,p)//PCM/M052X/6-31G(d) calculations.
The energies have been homogenously decreased by 0.45 eV, which is the difference between
the experimental absorption maximum of adenine and the vertical absorption energy computed at
the adopted level of theory. In dashes: calculated differential absorption spectra corresponding to
the disappearance of reacting adenines and the formation of I (red) and A=A and AA* (black,
assuming that their yields are roughly equal, in line with literature6); the oscillator strength is
proportional to the spectral area; the spectral width is 0.4 eV. (B) Spectrum obtained for (dA)20 at
20 ms (red) compared to the difference of steady-state absorption spectra recorded after and
before irradiation (black). The spectra are normalized by the concentration of absorbed photons
[h].
The computed absorption spectrum of I (Fig. SI-7) exhibits two transitions located at
energies lower than those of both the reacting adenines (AA) and the dimers A=A and
AA* by at least 0.25 and 0.6 eV (Fig. 3A). This finding suggests that, at wavelengths
longer than 300 nm, the I spectrum is more intense compared to those of the reactants
and the final photoproducts. On this ground, we searched the fingerprints of I in the
time-resolved spectra recorded by laser flash photolysis. This technique has been applied
7
successfully to study the formation of 64PPs in thymine single strands from the oxetane
intermediate.16
The studied adenine eicosamers (dA)20 (HPLC purified from Eurogentec Europe) were
dissolved in phosphate buffer (0.15 mol∙L-1 NaH2PO4, 0.15 mol∙L-1 Na2HPO4). The
solutions were excited at 266 nm with 5 ns pulses. We stress that such experiments are
very delicate and specific experimental protocols, preventing excitation of UV-damaged
oligomers are required (see SI). The average power used in our experiments varied from
0.5 to 2x106 W∙cm-2. Under such experimental conditions, in addition to UV-induced
dimerization, ionization processes give rise to adenine radicals. 17The typical absorption
bands of the adenine radical are easily distinguishable in the (dA)20 spectrum recorded at
1.7 µs. This is shown in Fig. 4A together with the spectrum of the deprotonated radical
cation reported by Candeias and Steenken.18 The intensity of the band at 600 nm varies
linearly with the concentration of ejected hydrated electrons [e -] (Fig. SI-8), determined
from their absorption band peaking at 720 nm.17, 19 This is not the case for the UV band,
whose shape changes with the intensity of the excitation pulse. When the ratio of ejected
electrons versus absorbed photons [e-]/[h] drops from 1.5x10-3 to 0.3x10-3, we can
clearly distinguish a peak at 300 nm while the radical peak at 335 nm appears only as a
shoulder (inset in Fig. 4A).
8
Figure 4. Time-resolved absorption spectra (circles) obtained for (dA)20 at 1.7 µs (A) and 20 ms
(B) following laser excitation at 266 nm. The grey line in A corresponds to the spectrum of the
adenine radical (from reference 18; in arbitrary units). Inset in A: normalized spectra obtained for
different ratios of ejected electron versus absorbed photons; 1.5x10-3 (yellow) and 0.3x10-3
(cyan). Inset in B: dependence of the transient absorbance at 340 nm and 20ms on the
concentration of absorbed photons.
At much longer times, when the adenine radicals have disappeared, we observe a large
absorption band peaking at 300 nm with a weak tail extending to 420 nm (Fig. 4B). In
contrast to the species issued from electron ejection, the intensity of the band at 20 ms
scales with the concentration of absorbed photons (inset in Fig. 4B). The transient spectra
obtained for aerated and Argon bubbled solutions are practically the same. These
observations indicate that the chemical species responsible for the 300 nm band, which
appears at times shorter than the time resolution of the experimental setup (50 ns; Fig. SI-
9
9) originates from a singlet excited state. We have not observed any significant decay
within 100 ms. Beyond this time, the instability of the detector hinders the measurements.
The spectrum at 20 ms is compared in Fig. 3B with the difference of the steady-state
spectra obtained before and after 266 nm irradiation. The same differential spectrum is
obtained using either a continuous light source or laser pulses of varying energy. In all
cases, the spectrum intensity scales with the number of absorbed photons (Fig. SI-10) and
it is not altered for several hours after irradiation; from these steady-state spectra and
using the average molar absorption coefficient of the adenine dimers reported by
Pörschke for (dA)2,1 we estimated that the dimerization quantum yield is 7x10-3, which
of the same order of magnitude as that reported by Clingen and Davies for poly(dA)
(3x10-3).6
The comparison of the two spectra in Fig. 3B shows that spectral changes occur between
100 ms and 5 min, which is the time between irradiation and recording of the steady-state
spectrum. The peak shifts from 300 to 295 nm and the intensity between 295 and 400 nm
decreases significantly. These modifications compare nicely with the differences in the
corresponding theoretical spectra (Fig. 3A), computed assuming that the two final
photoproducts are formed with the same quantum yield.
In view of the above discussion, we assign the transient spectrum to the precursor I. Its
conversion to A=A and AA* is obviously slower compared with the evolution of oxetane to
64PPs in thymine strands, which takes place within 4 ms, suggesting that the stability of the
studied azetidine intermediate is larger than that ofthe oxetane.16 The stability of four-ring
dimerization precursors has been correlated with the electronegativity of the involved
heteroatoms. In the presence of sulphur atom, which is less electronegative than nitrogen and
10
oxygen atoms, the reaction intermediate thietane is so stable that it has been isolated and
characterized.20 Thus, it is not surprising that the Nitrogen containing precursor I, is less stable
than thietane but longer lived than oxetane.
In conclusion, we studied adenine dimerization combining theoretical tools (molecular
dynamics simulations and quantum mechanical methods) with steady-state and timeresolved absorption spectroscopy. We identified a reaction intermediate which is formed
from a singlet excited state and characterized by a lifetime comprised between 0.1 s and a
few minutes. We showed that the photodimerization reaction is governed by
conformational equilibria.Different interbase stacking geometries lead to different
patterns of bonding/non-bonding interactions between atoms of the two adenines, and,
therefore, determinewhich photodimers can be potentially formed without a significant
energy barrier. Moreover, some of the possible photoproducts do not correspond to stable
minima, as shown by our results on (AA)syn-syn, or they are less stable than the reactants.
Consequently, adenine photodimerization is possible only for syn-anti conformers. This
finding explains why base pairing,dramatically destabilizing syn conformers,diminishes
drastically the quantum yield of the UV-induced adenine dimers.6It would be interesting
to perform the same type of investigation for other photodimerization reactions, not only
inDNA strands but also in other multichromophoric systems whose geometry is
characterized by many degrees of freedom.
Supporting Information:Computational Details, Additional Computational Results,
Experimental Details, Additional Experimental Results, Cartesian Coordinates.
11
AUTHOR INFORMATION
Corresponding Authors
[email protected]
[email protected]
The authors declare no competing financial interests.
ACKNOWLEDGMENT
Financial support from the French Agency for Research (ANR-10-BLAN-0809-01), the CNRSCNR PICS project (N°6827-2015)/Bilateral CNR/CNRS and the Legge Regionale 5 Campania
2007 is acknowledged.
12
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